Autoreactivity and Exceptional CDR Plasticity (but Not Unusual Polyspecificity) Hinder Elicitation of the Anti-HIV Antibody 4E10

The broadly-neutralizing anti-HIV antibody 4E10 recognizes an epitope in the membrane-proximal external region of the HIV envelope protein gp41. Previous attempts to elicit 4E10 by vaccination with envelope-derived or reverse-engineered immunogens have failed. It was presumed that the ontogeny of 4E10-equivalent responses was blocked by inherent autoreactivity and exceptional polyreactivity. We generated 4E10 heavy-chain knock-in mice, which displayed significant B cell dysregulation, consistent with recognition of autoantigen/s by 4E10 and the presumption that tolerance mechanisms may hinder the elicitation of 4E10 or 4E10-equivalent responses. Previously proposed candidate 4E10 autoantigens include the mitochondrial lipid cardiolipin and a nuclear splicing factor, 3B3. However, using carefully-controlled assays, 4E10 bound only weakly to cardiolipin-containing liposomes, but also bound negatively-charged, non-cardiolipin-containing liposomes comparably poorly. 4E10/liposome binding was predominantly mediated by electrostatic interactions rather than presumed hydrophobic interactions. The crystal structure of 4E10 free of bound ligands showed a dramatic restructuring of the combining site, occluding the HIV epitope binding site and revealing profound flexibility, but creating an electropositive pocket consistent with non-specific binding of phospholipid headgroups. These results strongly suggested that antigens other than cardiolipin mediate 4E10 autoreactivity. Using a synthetic peptide library spanning the human proteome, we determined that 4E10 displays limited and focused, but unexceptional, polyspecificity. We also identified a novel autoepitope shared by three ER-resident inositol trisphosphate receptors, validated through binding studies and immunohistochemistry. Tissue staining with 4E10 demonstrated reactivity consistent with the type 1 inositol trisphosphate receptor as the most likely candidate autoantigen, but is inconsistent with splicing factor 3B3. These results demonstrate that 4E10 recognition of liposomes competes with MPER recognition and that HIV antigen and autoepitope recognition may be distinct enough to permit eliciting 4E10-like antibodies, evading autoimmunity through directed engineering. However, 4E10 combining site flexibility, exceptional for a highly-matured antibody, may preclude eliciting 4E10 by conventional immunization strategies

Protein interaction mapping with ribosome-displayed using PLATO ORF libraries

Identifying physical interactions between proteins and other molecules is a critical aspect of biological analysis. Here we describe PLATO, an in vitro method for mapping such interactions by affinity enrichment of a library of full-length open reading frames displayed on ribosomes, followed by massively parallel analysis using DNA sequencing. We demonstrate the broad utility of the method by identifying known and new interacting partners of LYN kinase, patient autoantibodies and the small molecules gefitinib and dasatinib

Long-adapter single-strand oligonucleotide probes for the massively multiplexed cloning of kilobase genome regions

As the catalogue of sequenced genomes and metagenomes continues to grow, massively parallel approaches for the comprehensive and functional analysis of gene products and regulatory elements are becoming increasingly valuable. Current strategies to synthesize or clone complex libraries of DNA sequences are limited by the length of the DNA targets, throughput and cost. Here, we show that long-adapter single-strand oligonucleotide (LASSO) probes can capture and clone thousands of kilobase DNA fragments in a single reaction. As a proof-of-principle, we simultaneously cloned >3,000 bacterial open reading frames (ORFs) from E. coli genomic DNA (spanning 400–5,000 bp targets). Targets were enriched up to a median of ~60-fold compared to non-targeted genomic regions. At a cutoff of 3 times the median non-target reads per kilobase of genetic element per million reads, ~75% of the targeted ORFs were successfully captured. We also show that LASSO probes can clone human ORFs from complementary DNA, and an ORF library from a human-microbiome sample. LASSO probes could be used for the preparation of long-read sequencing libraries and for massively multiplexed cloning

Ontogeny of Recognition Specificity and Functionality for the Broadly Neutralizing Anti-HIV Antibody 4E10

The process of antibody ontogeny typically improves affinity, on-rate, and thermostability, narrows polyspecificity, and rigidifies the combining site to the conformer optimal for binding from the broader ensemble accessible to the precursor. However, many broadly-neutralizing anti-HIV antibodies incorporate unusual structural elements and recognition specificities or properties that often lead to autoreactivity. The ontogeny of 4E10, an autoreactive antibody with unexpected combining site flexibility, was delineated through structural and biophysical comparisons of the mature antibody with multiple potential precursors. 4E10 gained affinity primarily by off-rate enhancement through a small number of mutations to a highly conserved recognition surface. Controverting the conventional paradigm, the combining site gained flexibility and autoreactivity during ontogeny, while losing thermostability, though polyspecificity was unaffected. Details of the recognition mechanism, including inferred global effects due to 4E10 binding, suggest that neutralization by 4E10 may involve mechanisms beyond simply binding, also requiring the ability of the antibody to induce conformational changes distant from its binding site. 4E10 is, therefore, unlikely to be re-elicited by conventional vaccination strategies

Application of a synthetic human proteome to autoantigen discovery through PhIP-Seq

In this study, we improve on current autoantigen discovery approaches by creating a synthetic representation of the complete human proteome, the T7 “peptidome” phage display library (T7-Pep), and use it to profile the autoantibody repertoires of individual patients. We provide methods for 1) designing and cloning large libraries of DNA microarray-derived oligonucleotides encoding peptides for display on bacteriophage, and 2) analysis of the peptide libraries using high throughput DNA sequencing. We applied phage immunoprecipitation sequencing (PhIP-Seq) to identify both known and novel autoantibodies contained in the spinal fluid of three patients with paraneoplastic neurological syndromes. We also show how our approach can be used more generally to identify peptide-protein interactions and point toward ways in which this technology will be further developed in the future. We envision that PhIP-Seq can become an important new tool in autoantibody analysis, as well as proteomic research in general

LINE-1 ORF2p expression is nearly imperceptible in human cancers

Background Long interspersed element-1 (LINE-1, L1) is the major driver of mobile DNA activity in modern humans. When expressed, LINE-1 loci produce bicistronic transcripts encoding two proteins essential for retrotransposition, ORF1p and ORF2p. Many types of human cancers are characterized by L1 promoter hypomethylation, L1 transcription, L1 ORF1p protein expression, and somatic L1 retrotransposition. ORF2p encodes the endonuclease and reverse transcriptase activities required for L1 retrotransposition. Its expression is poorly characterized in human tissues and cell lines. Results We report mass spectrometry-based tumor proteome profiling studies wherein ORF2p eludes detection. To test whether ORF2p could be detected with specific reagents, we developed and validated five rabbit monoclonal antibodies with immunoreactivity for specific epitopes on the protein. These reagents readily detect ectopic ORF2p expressed from bicistronic L1 constructs. However, endogenous ORF2p is not detected in human tumor samples or cell lines by western blot, immunoprecipitation, or immunohistochemistry despite high levels of ORF1p expression. Moreover, we report endogenous ORF1p-associated interactomes, affinity isolated from colorectal cancers, wherein we similarly fail to detect ORF2p. These samples include primary tumors harboring hundreds of somatically acquired L1 insertions. The new data are available via ProteomeXchange with identifier PXD013743. Conclusions Although somatic retrotransposition provides unequivocal genetic evidence for the expression of ORF2p in human cancers, we are unable to directly measure its presence using several standard methods. Experimental systems have previously indicated an unequal stoichiometry between ORF1p and ORF2p, but in vivo, the expression of these two proteins may be more strikingly uncoupled. These findings are consistent with observations that ORF2p is not tolerable for cell growth

Comprehensive profiling of pre-infection antibodies identifies HIV targets associated with viremic control and viral load

BackgroundHigh HIV viral load (VL) is associated with increased transmission risk and faster disease progression. HIV controllers achieve viral suppression without antiretroviral (ARV) treatment. We evaluated viremic control in a community-randomized trial with >48,000 participants.MethodsA massively multiplexed antibody profiling system, VirScan, was used to quantify pre- and post-infection antibody reactivity to HIV peptides in 664 samples from 429 participants (13 controllers, 135 viremic non-controllers, 64 other non-controllers, 217 uninfected persons). Controllers had VLs <2,000 copies/mL with no ARV drugs detected at the first HIV-positive visit and one year later. Viremic non-controllers had VLs 2,000 copies/mL with no ARV drugs detected at the first HIV-positive visit. Other non-controllers had either ARV drugs detected at the first HIV-positive visit (n=47) or VLs <2,000 copies/mL with no ARV drugs detected at only one HIV-positive visit (n=17).ResultsWe identified pre-infection HIV antibody reactivities that correlated with post-infection VL. Pre-infection reactivity to an epitope in the HR2 domain of gp41 was associated with controller status and lower VL. Pre-infection reactivity to an epitope in the C2 domain of gp120 was associated with non-controller status and higher VL. Different patterns of antibody reactivity were observed over time for these two epitopes.ConclusionThese studies suggest that pre-infection HIV antibodies are associated with controller status and modulation of HIV VL. These findings may inform research on antibody-based interventions for HIV treatment

Measles virus infection diminishes preexisting antibodies that offer protection from other pathogens

Measles virus is directly responsible for more than 100,000 deaths yearly. Epidemiological studies have associated measles with increased morbidity and mortality for years after infection, but the reasons why are poorly understood. Measles virus infects immune cells, causing acute immune suppression. To identify and quantify long-term effects of measles on the immune system, we used VirScan, an assay that tracks antibodies to thousands of pathogen epitopes in blood. We studied 77 unvaccinated children before and 2 months after natural measles virus infection. Measles caused elimination of 11 to 73% of the antibody repertoire across individuals. Recovery of antibodies was detected after natural reexposure to pathogens. Notably, these immune system effects were not observed in infants vaccinated against MMR (measles, mumps, and rubella), but were confirmed in measles-infected macaques. The reduction in humoral immune memory after measles infection generates potential vulnerability to future infections, underscoring the need for widespread vaccination.Peer reviewe

Applications of VirScan to broad serological profiling of bat reservoirs for emerging zoonoses

IntroductionBats are important providers of ecosystem services such as pollination, seed dispersal, and insect control but also act as natural reservoirs for virulent zoonotic viruses. Bats host multiple viruses that cause life-threatening pathology in other animals and humans but, themselves, experience limited pathological disease from infection. Despite bats’ importance as reservoirs for several zoonotic viruses, we know little about the broader viral diversity that they host. Bat virus surveillance efforts are challenged by difficulties of field capture and the limited scope of targeted PCR- or ELISA-based molecular and serological detection. Additionally, virus shedding is often transient, thus also limiting insights gained from nucleic acid testing of field specimens. Phage ImmunoPrecipitation Sequencing (PhIP-Seq), a broad serological tool used previously to comprehensively profile viral exposure history in humans, offers an exciting prospect for viral surveillance efforts in wildlife, including bats.MethodsHere, for the first time, we apply PhIP-Seq technology to bat serum, using a viral peptide library originally designed to simultaneously assay exposures to the entire human virome.ResultsUsing VirScan, we identified past exposures to 57 viral genera—including betacoronaviruses, henipaviruses, lyssaviruses, and filoviruses—in semi-captive Pteropus alecto and to nine viral genera in captive Eonycteris spelaea. Consistent with results from humans, we find that both total peptide hits (the number of enriched viral peptides in our library) and the corresponding number of inferred past virus exposures in bat hosts were correlated with poor bat body condition scores and increased with age. High and low body condition scores were associated with either seropositive or seronegative status for different viruses, though in general, virus-specific age-seroprevalence curves defied assumptions of lifelong immunizing infection, suggesting that many bat viruses may circulate via complex transmission dynamics.DiscussionOverall, our work emphasizes the utility of applying biomedical tools, like PhIP-Seq, first developed for humans to viral surveillance efforts in wildlife, while highlighting opportunities for taxon-specific improvements

Evaluation of multi-assay algorithms for cross-sectional HIV incidence estimation in settings with universal antiretroviral treatment.

BACKGROUND: Multi-assay algorithms (MAAs) are used to estimate population-level HIV incidence and identify individuals with recent infection. Many MAAs use low viral load (VL) as a biomarker for long-term infection. This could impact incidence estimates in settings with high rates of early HIV treatment initiation. We evaluated the performance of two MAAs that do not include VL. METHODS: Samples were collected from 219 seroconverters (infected  1 year) in the HPTN 071 (PopART) trial; 28.8% of seroconverter samples and 73.2% of non-seroconverter samples had VLs ≤ 400 copies/mL. Samples were tested with the Limiting Antigen Avidity assay (LAg) and JHU BioRad-Avidity assays. Antibody reactivity to two HIV peptides was measured using the MSD U-PLEX assay. Two MAAs were evaluated that do not include VL: a MAA that includes the LAg-Avidity assay and BioRad-Avidity assay (LAg + BR) and a MAA that includes the LAg-Avidity assay and two peptide biomarkers (LAg + PepPair). Performance of these MAAs was compared to a widely used MAA that includes LAg and VL (LAg + VL). RESULTS: The incidence estimate for LAg + VL (1.29%, 95% CI: 0.97-1.62) was close to the observed longitudinal incidence (1.34% 95% CI: 1.17-1.53). The incidence estimates for the other two MAAs were higher (LAg + BR: 2.56%, 95% CI 2.01-3.11; LAg + PepPair: 2.84%, 95% CI: 1.36-4.32). LAg + BR and LAg + PepPair also misclassified more individuals infected > 2 years as recently infected than LAg + VL (1.2% [42/3483 and 1.5% [51/3483], respectively, vs. 0.2% [6/3483]). LAg + BR classified more seroconverters as recently infected than LAg + VL or LAg + PepPair (80 vs. 58 and 50, respectively) and identified ~ 25% of virally suppressed seroconverters as recently infected. CONCLUSIONS: The LAg + VL MAA produced a cross-sectional incidence estimate that was closer to the longitudinal estimate than two MAAs that did not include VL. The LAg + BR MAA classified the greatest number of individual seroconverters as recently infected but had a higher false recent rate